This project has the basic research purpose of elucidating how gamma-H2AX focal formation at DNA DSB sites help maintain genome stability. There are related translational purposes, including the use of H2AX phosphorylation as a biological dosimeter used in conjunction with radiation treatment, as an indicator of the ability of an unknown compund to cause DNS DSBs and hence be a potential drug for cancer treatment, and as an indicator of the efficicy of a potential in humans such as during Phase 0 protocols. The major advance published during this fiscal year has been the demonstration of the bysrtander effect in human tissue artificial models. Artificial tissues reconstruct the structure, differentiation, and metabolic interactions of normal tissues in the body. We examined the characteristcs of the bystander effect in two artificial human tissues, airway and full thickness skin by subjecting a thin plane of cells through the tissue to alpha-particle radiation using a focused microbeam. Bystander cells away from the irradiation plane were compared to cells in the irradiation plane for a variety of biological end points during seven days post-irradiation. In marked contrast to DNA DSB dynamics in irradiated cells, in which DSB formation is maximal by 30 min after irradiation, the incidence of DSBs in bystander cells gradually increased to a maximum 12 to 48 h after irradiation and then decreased over the seven-day time course. At the maxima, 40% to 60% of the cells in the bystander regions were affected, a 4- to 6-fold increase over controls. These increases in bystander DSB formation were followed by increased levels of apoptosis and micronucleus formation, by loss of nuclear DNA methylation, and by an increased fraction of senescent cells. These findings demonstrate the extent of involvement of DNA DSBs in bystander responses in artificial 3D human tissues and support the notion that bystander DNA DSBs are precursors to widespread downstream effects in these tissues. These findings suggest that the bystander response may be an important factor in the response of animals including humans to ionizing radiation. Understanding this response and how to control it may offer benefits to patients undergoing radiation therapy by lessening off-target side effects. In addition, bystander cells exhibiting postirradiation signs of genomic instability may be more prone than unaffected cells to become cancerous. Thus, this study points to the importance of considering the indirect biological effects of radiation in cancer risk assessment.